NIST logo
*

Bitter Orange Standard Reference Materials

Summary:


Three bitter orange-containing Standard Reference Materials have been developed as part of the Chemical Science Division's program to produce SRMs related to dietary supplements. Levels of five citrus alkaloids and caffeine were determined, and the materials were screened for pesticide residues and toxic heavy metals. Multiple analytical methods with measurements made at NIST and by collaborating laboratories were used to assign values. The methods are independent, with two types of extractions, two separation methods, and four detection methods. The bitter orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods.

Description:


The development of bitter orange-containing dietary supplement SRMs is part of a collaborative effort between NIST and the National Institutes of Health, Office of Dietary Supplements (NIH-ODS). In December 2003 the Food and Drug Administration ruled that dietary supplements that contain ephedrine alkaloids are adulterated, based on potential health risks associated with their use. This ruling, which became effective in April 2004, prohibits the sale of ephemera-containing dietary supplements in the United States. As a result of this action, many manufacturers of dietary supplements reformulated weight loss products with ingredients that are considered to be safer alternatives to ephedra, such as bitter orange, and reference materials are needed to support Good Manufacturing Practices (GMPs) for these products.

Additional Technical Details:


As with other complex-matrix reference materials produced by NIST, value assignment of the bitter orange SRMs was accomplished by using multiple independent analytical methods with measurements performed at NIST and by collaborating laboratories. Data from the different methods and sources were evaluated statistically and combined to yield a consensus value and expanded uncertainty interval. As many as six data sets were utilized in value assignment of the citrus alkaloids. The corresponding methods are substantially independent; at NIST at least two extraction approaches, two chromatographic separation methods, and four modes of detection were used. Extraction studies were carried out to develop approaches for quantitative recovery of the bitter orange alkaloids from the various types of samples. Three extraction methods were studied: sonication extraction, pressurized-fluid extraction, and Soxhlet extraction. In general, higher levels of the alkaloids were extracted with longer solvent contact times, particularly for the ground plant material. Multiple extraction cycles were also useful in maximizing analyte recovery. Instrumental analytical methods included the use of ion-pairing liquid chromatography with fluorescence detection, and liquid chromatography with a pentafluorophenyl column in combination with tandem mass spectrometry. These robust methods offered substantial independence, and provide enhanced sensitivity and selectivity resulting in accurate and precise measurements.  

Photograph of packets of SRM 3258 Bitter Orange Fruit

Major Accomplishments:

  • Multiple analytical methods have been developed for the measurement of citrus alkaloids in plant, extract, and finished product samples.
  • Three bitter orange containing SRMs have been produced with certified, reference, or information values for citrus alkaloids and toxic elements and a certified value for caffeine (in one SRM):

SRM 3258 Bitter Orange (Fruit)
SRM 3259 Bitter Orange Extract
SRM 3260 Bitter Orange-Containing Solid Oral Dosage Form
SRM 3261 Bitter Orange Dietary Supplement Suite.

Click on an SRM name to be transferred to a page where you can view its Certificate of Analysis. 

Start Date:

October 1, 2002

End Date:

April 3, 2008

Lead Organizational Unit:

mml

Staff:

Mary Bedner
Karen E. Murphy
Bryant Nelson
Catherine A. Rimmer
Michele M. Schantz
Jeanice B. Thomas
Laura J. Wood 

Associated Products:

Putzbach, K., Sharpless, K.E., and Sander, L.C., Determination of Bitter Orange Alkaloids in Dietary Supplements Standard Reference Materials by Liquid Chromatography with Ultraviolet Absorbance and Fluorescence Detection, J. Chromatogr., 1156:304-311 (2007).

Putzbach, K., Rimmer, C.A., Sharpless, K.E., Wise, S.A., and Sander, L.C., Determination of Bitter Orange Alkaloids in Dietary Supplement Standard Reference Materials by Liquid Chromatography with Atmospheric-Pressure Ionization Mass Spectrometry, Anal. Bioanal. Chem., 389:197-205 (2007).

Nelson, B.C., Putzbach, K., Sharpless, K.E., and Sander, L.C., “Mass Spectrometric Determination of the Predominant Adrenergic Alkaloids in Bitter Orange (Citrus aurantium) J. Agric. Food Chem., 55:9769-9775 (2007).

Santana, J., Sharpless, K.E., and Nelson, B.C., "Liquid Chromatographic Methods for the Determination of Para-Synephrine and Meta-Synephrine Positional Isomers in Bitter Orange-Containing Dietary Supplements," Food Chem., 109: 675-682 (2008).

Sander, L.C., Putzbach, K., Nelson, B.C., Rimmer, C., Bedner, M., Brown Thomas, J., Porter, B.J., Wood, L.J., Schantz, M.M., Sharpless, K.E., Wise, S.A., Yen, J.H., Siitonen, P.H., NguyenPho, A., Roman, M.C., Betz, J.M., and Murphy, K.E., “Certification of Standard Reference Materials Containing Bitter Orange,” Anal. Bioanal. Chem., 391:2023-2034 (2008).

Contact

Lane C. Sander
Phone 301-975-3117
lane.sander@nist.gov

Katherine E. Sharpless
Phone 301-975-3121
katherine.sharpless@nist.gov

Stephen A. Wise
Phone 301-975-3112
stephen.wise@nist.gov